Human illness results from the complex interactions of integrated processes and factors, including genetic predispositions and environmental agents. The environmental genome project (EGP) was formally initiated to systematically and comprehensively evaluate how genetic polymorphisms impact our susceptibility to environmentally founded disease. The EGP has identified eight categories of environmentally responsive genes (ERG) that have been shown to react to environmental agents. These categories include cell cycle, DNA repair, cell division, cell signaling, cell structure, gene expression, apoptosis and metabolism. The genome is under continuous assault by a combination of both environmental and endogenous DNA damaging agents requiring a complex set of DNA repair proteins to resolve these genetic insults. However, there are clear inter-individual differences between humans in their susceptibility to DNA damaging agents that result from either pre-existing environmental exposures or genetic factors such as sequence variation or single- nucleotide polymorphisms, SNPs. Evaluating the functional impact of individual polymorphisms will require novel approaches and new reagents. During the first phase of this proposal, we will develop and characterize a series of isogenic DNA glycosylase deficient human cell lines for future studies towards evaluation of the functional significance of DNA repair gene SNPs and genetic variants in human cells. These studies are designed to provide essential reagents to aid in understanding the biological significance of human DNA polymorphisms and the role of these SNPs either alone or in combination with specific environmental stressors in disease outcomes as varied as cancer, aging-related disorders, stroke and diabetes. Upon successful completion of the 1st phase, we intend to have demonstrated the feasibility of producing stable human cell lines with complete deficiency in DNA repair proteins, specifically DNA glycosylases. Further, we propose to characterize each newly developed cell line with respect to mRNA and protein expression and DNA glycosylase activity and finally, each will be evaluated for the impact of the depletion of a single DNA repair gene product on the global transcriptome. The development of such isogenic human cells for an additional 140 DNA repair genes will be the topic of the second phase of this proposal, covering genes involved in Base Excision Repair (BER), Direct Reversal of Damage, Mismatch excision repair (MMR), Nucleotide Excision Repair (NER), Homologous Recombination, Non- homologous end-joining, the modulation of nucleotide pools, DNA polymerases, editing and processing nucleases, the Rad6 pathway, Chromatin Structure, DNA repair genes defective in diseases and conserved DNA damage response genes. PUBLIC HEALTH RELEVANCE: The over all goal of the phase I project is to develop cell lines each depleted of the known DNA repair associated glycosylases. In the proposal we plan to develop real time in vivo assays to monitor glycosylase activity. Additionally we intend to determine the effect of depletion of a single glycosylases on the global transcriptome.